Astrocytes form large gap junctional networks that contribute to ion and neurotransmitter homeostasis. Astrocytes concentrate in the lateral superior olive (LSO), a prominent auditory brainstem center. Compared to the LSO, astrocyte density is lower in the region dorsal to the LSO (dLSO) and in the internuclear space between the LSO, the superior paraolivary nucleus (SPN). We questioned whether astrocyte networks exhibit certain properties that reflect the precise neuronal arrangement. Employing whole-cell patch-clamp and concomitant injection of a gap junction-permeable tracer, we analyzed size and orientation of astrocyte networks in LSO, dLSO, and SPN-LSO in acute brainstem slices of mice at postnatal days 10-20. The majority of LSO networks exhibited an oval topography oriented orthogonally to the tonotopic axis, whereas dLSO networks showed no preferred orientation. This correlated with the overall astrocyte morphology in both regions, i.e. LSO astrocyte processes were oriented mainly orthogonally to the tonotopic axis. To assess the spread of small ions within LSO networks, we analyzed the diffusion of Na(+) signals between cells using Na(+) imaging. We found that Na(+) not only diffused between SR101(+) astrocytes, but also from astrocytes into SR101(-) cells. Using PLP-GFP mice for tracing, we could show that LSO networks contained astrocytes and oligodendrocytes. Together, our results demonstrate that LSO astrocytes and LSO oligodendrocytes form functional anisotropic panglial networks that are oriented predominantly orthogonally to the tonotopic axis. Thus, our results point toward an anisotropic ion and metabolite diffusion and a limited glial crosstalk between neighboring isofrequency bands in the LSO. GLIA 2016;64:1892-1911.

Astrocytes and oligodendrocytes in different brain regions form panglial networks and the topography of such networks can correlate with neuronal topography and function. Astrocyte-oligodendrocyte networks in the lateral superior olive (LSO)—an auditory brainstem nucleus—were found to be anisotropic with a preferred orientation orthogonally to the tonotopic axis. We hypothesized that such a specialization might be present in other tonotopically organized brainstem nuclei, too. Thus, we analyzed gap junctional coupling in the center of the inferior colliculus (IC)—another nucleus of the auditory brainstem that exhibits tonotopic organization. In acute brainstem slices obtained from mice, IC networks were traced employing whole-cell patch-clamp recordings of single sulforhodamine (SR) 101-identified astrocytes and concomitant intracellular loading of the gap junction-permeable tracer neurobiotin. The majority of dye-coupled networks exhibited an oval topography, which was preferentially oriented orthogonal to the tonotopic axis. Astrocyte processes showed preferentially the same orientation indicating a correlation between astrocyte and network topography. In addition to SR101-positive astrocytes, IC networks contained oligodendrocytes. Using Na+ imaging, we analyzed the capability of IC networks to redistribute small ions. Na+ bi-directionally diffused between SR101-positive astrocytes and SR101-negative cells—presumably oligodendrocytes—showing the functionality of IC networks. Taken together, our results demonstrate that IC astrocytes and IC oligodendrocytes form functional anisotropic panglial networks that are preferentially oriented orthogonal to the tonotopic axis. Thus, our data indicate that the topographic specialization of glial networks seen in IC and LSO might be a general feature of tonotopically organized auditory brainstem nuclei.

Neuronal inhibition is mediated by glycine and/or GABA. Inferior colliculus (IC) neurons receive glycinergic and GABAergic inputs, whereas inhibition in hippocampus (HC) predominantly relies on GABA. Astrocytes heterogeneously express neurotransmitter transporters and are expected to adapt to the local requirements regarding neurotransmitter homeostasis. Here we analyzed the expression of inhibitory neurotransmitter transporters in IC and HC astrocytes using whole-cell patch-clamp and single-cell reverse transcription-PCR. We show that most astrocytes in both regions expressed functional glycine transporters (GlyTs). Activation of these transporters resulted in an inward current (IGly) that was sensitive to the competitive GlyT1 agonist sarcosine. Astrocytes exhibited transcripts for GlyT1 but not for GlyT2. Glycine did not alter the membrane resistance (RM) arguing for the absence of functional glycine receptors (GlyRs). Thus, IGly was mainly mediated by GlyT1. Similarly, we found expression of functional GABA transporters (GATs) in all IC astrocytes and about half of the HC astrocytes. These transporters mediated an inward current (IGABA) that was sensitive to the competitive GAT-1 and GAT-3 antagonists NO711 and SNAP5114, respectively. Accordingly, transcripts for GAT-1 and GAT-3 were found but not for GAT-2 and BGT-1. Only in hippocampal astrocytes, GABA transiently reduced RM demonstrating the presence of GABAA receptors (GABAARs). However, IGABA was mainly not contaminated by GABAAR-mediated currents as RM changes vanished shortly after GABA application. In both regions, IGABA was stronger than IGly. Furthermore, in HC the IGABA/IGly ratio was larger compared to IC. Taken together, our results demonstrate that astrocytes are heterogeneous across and within distinct brain areas. Furthermore, we could show that the capacity for glycine and GABA uptake varies between both brain regions.Electronic supplementary materialThe online version of this article (10.1186/s13041-018-0346-y) contains supplementary material, which is available to authorized users.

The α2δ3 auxiliary subunit of voltage-activated calcium channels is required for normal synaptic transmission and precise temporal processing of sounds in the auditory brainstem. In mice its loss additionally leads to an inability to distinguish amplitude-modulated tones. Furthermore, loss of function of α2δ3 has been associated with autism spectrum disorder in humans. To investigate possible alterations of network activity in the higher-order auditory system in α2δ3 knockout mice, we analyzed neuronal activity patterns and topography of frequency tuning within networks of the auditory cortex (AC) using two-photon Ca2+ imaging. Compared to wild-type mice we found distinct subfield-specific alterations in the primary auditory cortex, expressed in overall lower correlations between the network activity patterns in response to different sounds as well as lower reliability of these patterns upon repetitions of the same sound. Higher AC subfields did not display these alterations but showed a higher amount of well-tuned neurons along with lower local heterogeneity of the neurons’ frequency tuning. Our results provide new insight into AC network activity alterations in an autism spectrum disorder-associated mouse model.

The α2δ3 auxiliary subunit of voltage-activated calcium channels is required for normal synaptic transmission and precise temporal processing of sounds in the auditory brainstem. In mice its loss additionally leads to an inability to distinguish amplitude-modulated (AM) tones. Furthermore, loss of function of α2δ3 has been associated with autism spectrum disorder (ASD) in humans. To investigate possible alterations of network activity in the high-order auditory system in α2δ3 KO mice, we analyzed neuronal activity patterns and topography of frequency tuning within networks of the auditory cortex (AC) using two-photon Ca2+ imaging. We found subfield-specific alterations, expressed in overall lower correlations between the network activity patterns in response to different sounds as well as lower reliability of these patterns upon repetitions of the same sound in the primary auditory cortex (A1). Higher AC subfields did not display these alterations but showed a higher amount of well-tuned neurons along with lower local heterogeneity of the neurons′ frequency tuning. Our results provide new insight into AC network activity alterations in an ASD-associated mouse model.